Design and Synthesis of AMPK Activators and GDF15 Inducers.

Targeting growth differentiation factor 15 (GDF15) is a recent strategy for the treatment of obesity and type 2 diabetes mellitus (T2DM). Here, we designed, synthesized, and pharmacologically evaluated in vitro a novel series of AMPK activators to upregulate GDF15 levels. These compounds were structurally based on the (1-dibenzylamino-3-phenoxy)propan-2-ol structure of the orphan ubiquitin E3 ligase subunit protein Fbxo48 inhibitor, BC1618. This molecule showed a better potency than metformin, increasing GDF15 mRNA levels in human Huh-7 hepatic cells. Based on BC1618, structural modifications have been performed to create a collection of diversely substituted new molecules. Of the thirty-five new compounds evaluated, compound 21 showed a higher increase in GDF15 mRNA levels compared with BC1618. Metformin, BC1618, and compound 21 increased phosphorylated AMPK, but only 21 increased GDF15 protein levels. Overall, these findings indicate that 21 has a unique capacity to increase GDF15 protein levels in human hepatic cells compared with metformin and BC1618.

Insights into the Pharmacokinetics and In Vitro Cell-Based Studies of the Imidazoline I2 Receptor Ligand B06.

The impact of neurodegenerative diseases (ND) is becoming unbearable for humankind due to their vast prevalence and the lack of efficacious treatments. In this scenario, we focused on imidazoline I2 receptors (I2-IR) that are widely distributed in the brain and are altered in patients with brain disorders. We took the challenge of modulating I2-IR by developing structurally new molecules, in particular, a family of bicyclic α-iminophosphonates, endowed with high affinity and selectivity to these receptors. Treatment of two murine models, one for age-related cognitive decline and the other for Alzheimer's disease (AD), with representative compound B06 ameliorated their cognitive impairment and improved their behavioural condition. Furthermore, B06 revealed beneficial in vitro ADME-Tox properties. The pharmacokinetics (PK) and metabolic profile are reported to de-risk B06 for progressing in the preclinical development. To further characterize the pharmacological properties of B06, we assessed its neuroprotective properties and beneficial effect in an in vitro model of Parkinson's disease (PD). B06 rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine (6-OHDA) and showed a crucial anti-inflammatory effect in a cellular model of neuroinflammation. This research reveals B06 as a putative candidate for advancing in the difficult path of drug discovery and supports the modulation of I2-IR as a fresh approach for the therapy of ND.

Effects of Preoperative Quadruple Therapy for Helicobacter pylori on Bariatric Surgery Metabolic Outcomes.

PURPOSE: To assess the effects of Helicobacter pylori (HP) eradication with an omeprazole, clarithromycin, amoxicillin, and metronidazole (OCAM) regimen on the metabolic profile and weight loss 12 months after bariatric surgery (BS). METHODS: Retrospective analysis of a prospective cohort of patients with morbid obesity undergoing BS. HP presence was tested preoperatively by gastric biopsy and treated with OCAM when positive. Short-term metabolic outcomes and weight loss were evaluated. RESULTS: HP infection was detected in 75 (45.7%) of the 164 patients included. OCAM effectiveness was 90.1%. HP-negative patients had a greater reduction in glucose levels at 3 (-14.6 ± 27.5 mg/dL HP-treated vs -22.0 ± 37.1 mg/dL HP-negative, p=0.045) and 6 months (-13.7 ± 29.4 mg/dL HP-treated vs -26.4 ± 42.6 mg/dL HP-negative, p= 0.021) and greater total weight loss (%TWL) at 6 (28.7 ± 6.7% HP-treated vs 30.45 ± 6.48% HP-negative, p= 0.04) and 12 months (32.21 ± 8.11% HP-treated vs 35.14 ± 8.63% HP-negative, p= 0.023). CONCLUSIONS: Preoperative treatment with OCAM has been associated to poorer glycemic and weight loss outcomes after BS. More research is needed on the influence of OCAM on gut microbiota, and in turn, the effect of the latter on metabolic and weight loss outcomes after BS.

Sex differences in the antidepressant-like response and molecular events induced by the imidazoline-2 receptor agonist CR4056 in rats.

In searching for novel targets to design antidepressants, among the characterized imidazoline receptors (IR), I2 receptors are an innovative therapeutical approach since they are dysregulated in major depressive disorder and by classical antidepressant treatments. In fact, several I2 agonists have been characterized for their antidepressant-like potential, but the results in terms of efficacy were mixed and exclusively reported in male rodents. Since there are well-known sex differences in antidepressant-like efficacy, this study characterized the potential effects induced by two I2 drugs, CR4056 (i.e., most promising drug already in phase II clinical trial for its analgesic properties) and B06 (a compound from a new family of bicyclic α-iminophosphonates) under the stress of the forced-swim test in male and female rats exposed to early-life stress. Moreover, some hippocampal neuroplasticity markers related to the potential effects observed were also evaluated (i.e., FADD, p-ERK/ERK, mBDNF, cell proliferation: Ki-67 + cells). The main results replicated the only prior study reporting the efficacy of CR4056 in male rats, while providing new data on its efficacy in females, which was clearly dependent on prior early-life stress exposure. Moreover, B06 showed no antidepressant-like effects in male or female rats. Finally, CR4056 increased FADD content and decreased cell proliferation in hippocampus, without affecting p-ERK/t-ERK ratio and/or mBDNF content. Interestingly, these effects were exclusively observed in female rats, and independently of early-life conditions, suggesting some distinctive molecular underpinnings participating in the therapeutic response of CR4056 for both sexes. In conjunction, these results present CR4056 with an antidepressant-like potential, especially in female rats exposed to stress early in life, together with some neuronal correlates described in the context of these behavioral changes in females.

Preclinical Evaluation of an Imidazole-Linked Heterocycle for Alzheimer's Disease.

Humanity is facing a vast prevalence of neurodegenerative diseases, with Alzheimer's disease (AD) being the most dominant, without efficacious drugs, and with only a few therapeutic targets identified. In this scenario, we aim to find molecular entities that modulate imidazoline I2 receptors (I2-IRs) that have been pointed out as relevant targets in AD. In this work, we explored structural modifications of well-established I2-IR ligands, giving access to derivatives with an imidazole-linked heterocycle as a common key feature. We report the synthesis, the affinity in human I2-IRs, the brain penetration capabilities, the in silico ADMET studies, and the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of this new bunch of I2-IR ligands. Selected compounds showed neuroprotective properties and beneficial effects in an in vitro model of Parkinson's disease, rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine, and showed crucial anti-inflammatory effects in a cellular model of neuroinflammation. After a preliminary pharmacokinetic study, we explored the action of our representative 2-(benzo[b]thiophen-2-yl)-1H-imidazole LSL33 in a mouse model of AD (5xFAD). Oral administration of LSL33 at 2 mg/Kg for 4 weeks ameliorated 5XFAD cognitive impairment and synaptic plasticity, as well as reduced neuroinflammation markers. In summary, this new I2-IR ligand that promoted beneficial effects in a well-established AD mouse model should be considered a promising therapeutic strategy for neurodegeneration.

Heterocycle-Based Multicomponent Reactions in Drug Discovery: From Hit Finding to Rational Design.

In the context of the structural complexity necessary for a molecule to selectively display a therapeutical action and the requirements for suitable pharmacokinetics, a robust synthetic approach is essential. Typically, thousands of relatively similar compounds should be prepared along the drug discovery process. In this respect, heterocycle-based multicomponent reactions offer advantages over traditional stepwise sequences in terms of synthetic economy, as well as the fast access to chemsets to study the structure activity relationships, the fine tuning of properties, and the preparation of larger amounts for preclinical phases. In this account, we briefly summarize the scientific methodology backing the research line followed by the group. We comment on the main results, clustered according to the targets and, finally, in the conclusion section, we offer a general appraisal of the situation and some perspectives regarding future directions in academic and private research.

An Imidazoline 2 Receptor Ligand Relaxes Mouse Aorta via Off-Target Mechanisms Resistant to Aging.

Imidazoline receptors (IR) are classified into three receptor subtypes (I1R, I2R, and I3R) and previous studies showed that regulation of I2R signaling has neuroprotective potential. In order to know if I2R has a role in modulating vascular tone in health and disease, we evaluated the putative vasoactive effects of two recently synthesized I2R ligands, diethyl (1RS,3aSR,6aSR)-5-(3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole -1-phosphonate (B06) and diethyl [(1-(3-chloro-4-fluorobenzyl)-5,5-dimethyl-4-phenyl-4,5-dihydro-1H-imidazol-4-yl]phosphonate] (MCR5). Thoracic aortas from Oncins France 1 (3- to 4-months-old) and C57BL/6 (3- to 4- and 16- to 17-months-old mice) were mounted in tissue baths to measure isometric tension. In young mice of both strains, MCR5 induced greater relaxations than either B06 or the high-affinity I2R selective ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI), which evoked marginal responses. MCR5 relaxations were independent of I2R, as IR ligands did not significantly affect them, involved activation of smooth muscle KATP channels and inhibition of L-type voltage-gated Ca2+ channels, and were only slightly modulated by endothelium-derived nitric oxide (negatively) and prostacyclin (positively). Notably, despite the presence of endothelial dysfunction in old mice, MCR5 relaxations were preserved. In conclusion, the present study provides evidence against a functional contribution of I2R in the modulation of vascular tone in the mouse aorta. Moreover, the I2R ligand MCR5 is an endothelium-independent vasodilator that acts largely via I2R-independent pathways and is resistant to aging. We propose MCR5 as a candidate drug for the management of vascular disease in the elderly.

Disease-modifying treatment with I2 imidazoline receptor ligand LSL60101 in an Alzheimer's disease mouse model: a comparative study with donepezil.

BACKGROUND AND PURPOSE: The development of effective therapeutic strategies against Alzheimer's disease (AD) remains a challenge. I2 imidazoline receptor ligands have a neuroprotective role in AD. Moreover, co-treatment of AChE inhibitors with neuroprotective agents have shown better effects on the prevention of dementia. Here, we assessed the potential therapeutic effect of the I2 ligand, donepezil and their combination in 5XFAD mice. EXPERIMENTAL APPROACH: 5XFAD female mice were treated with low doses (1 mg·kg-1 ·day-1 ) of LSL60101, donepezil and donepezil plus LSL60101, during 4 weeks per os. Novel object recognition, Morris water maze, open field, elevated plus maze and three-chamber tests were used to evaluate the cognitive and behavioural status after treatment. The effects on AD-like pathology were assessed with immunohistochemistry, western blot, ELISA and qPCR. KEY RESULTS: Chronic low-dose treatment with LSL60101 and donepezil reversed cognitive deficits and impaired social behaviour. LSL60101 treatment did not affect anxiety-like behaviour in contrast to donepezil. In the 5XFAD brains, LSL60101 and donepezil/LSL60101 treatments attenuated amyloid-ß pathology by decreasing amyloid-ß40 and amyloid-ß42 levels, amyloid-ß plaque number and tau hyperphosphorylation. These alterations were accompanied by reduced microglia marker Iba-1 levels and increased Trem2 gene expression. LSL60101 and donepezil decreased glial fibrillary acidic protein (GFAP) astrocytic marker reactivity. However, only LSL60101 and donepezil/LSL60101 treatments significantly increased the synaptic marker levels of post-synaptic density protein 95 and synaptophysin. CONCLUSION AND IMPLICATIONS: Chronic low-dose treatment with selective I2 - ligands can be an effective treatment for AD and provide insights into combination treatments for symptomatic and disease-modifying drugs.

I2 imidazoline receptor modulation protects aged SAMP8 mice against cognitive decline by suppressing the calcineurin pathway.

Brain aging and dementia are current problems that must be solved. The levels of imidazoline 2 receptors (I2-IRs) are increased in the brain in Alzheimer's disease (AD) and other neurodegenerative diseases. We tested the action of the specific and selective I2-IR ligand B06 in a mouse model of accelerated aging and AD, the senescence-accelerated mouse prone 8 (SAMP8) model. Oral administration of B06 for 4 weeks improved SAMP8 mouse behavior and cognition and reduced AD hallmarks, oxidative stress, and apoptotic and neuroinflammation markers. Likewise, B06 regulated glial excitatory amino acid transporter 2 and N-methyl-D aspartate 2A and 2B receptor subunit protein levels. Calcineurin (CaN) is a phosphatase that controls the phosphorylation levels of cAMP response element-binding (CREB), apoptotic mediator BCL-2-associated agonist of cell death (BAD) and GSK3ß, among other molecules. Interestingly, B06 was able to reduce the levels of the CaN active form (CaN A). Likewise, CREB phosphorylation, BAD gene expression, and other factors were modified after B06 treatment. Moreover, phosphorylation of a target of CaN, nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1), was increased in B06-treated mice, impeding the transcription of genes related to neuroinflammation and neural plasticity. In summary, this I2 imidazoline ligand can exert its beneficial effects on age-related conditions by modulating CaN pathway action and affecting several molecular pathways, playing a neuroprotective role in SAMP8 mice.

Benzofuranyl-2-imidazoles as imidazoline I2 receptor ligands for Alzheimer's disease.

Recent findings unveil the pharmacological modulation of imidazoline I2 receptors (I2-IR) as a novel strategy to face unmet medical neurodegenerative diseases. In this work, we report the chemical characterization, three-dimensional quantitative structure-activity relationship (3D-QSAR) and ADMET in silico of a family of benzofuranyl-2-imidazoles that exhibit affinity against human brain I2-IR and most of them have been predicted to be brain permeable. Acute treatment in mice with 2-(2-benzofuranyl)-2-imidazole, known as LSL60101 (garsevil), showed non-warning properties in the ADMET studies and an optimal pharmacokinetic profile. Moreover, LSL60101 induced hypothermia in mice while decreased pro-apoptotic FADD protein in the hippocampus. In vivo studies in the familial Alzheimer's disease 5xFAD murine model with the representative compound, revealed significant decreases in the protein expression levels of antioxidant enzymes superoxide dismutase and glutathione peroxidase in hippocampus. Overall, LSL60101 plays a neuroprotective role by reducing apoptosis and modulating oxidative stress.

Amelioration of BPSD-Like Phenotype and Cognitive Decline in SAMP8 Mice Model Accompanied by Molecular Changes after Treatment with I2-Imidazoline Receptor Ligand MCR5.

Behavioural and psychological symptoms of dementia (BPSD), including fear-anxiety- and depressive-like behaviour, are present in Alzheimer's disease (AD), together with memory decline. I2-imidazoline receptors (I2-IRs) have been associated with neuropsychiatric and neurodegenerative disorders, further, I2-IR ligands have demonstrated a neuroprotective role in the central nervous system (CNS). In this study, we assessed the effect of the I2-IR ligand MCR5 on both cognitive and non-cognitive symptoms in the Senescence accelerated mice prone 8 (SAMP8) mouse model. Oral administration of I2-IR ligand MCR5 (5 mg/kg/day for four weeks) in 10-month SAMP8 mice ameliorated both BPSD-like phenotype and cognitive decline by attenuating depressive-like behaviour, reducing fear-anxiety-like behaviour and improving cognitive performance using different tasks. Interaction of I2-IR ligand MCR5 with serotoninergic system did not account for behavioural or cognitive improvement, although changes in molecular pathways underlying depression and anxiety phenotype were observed. MCR5 increased levels of p-AKT, phosphorylated glycogen synthase kinase 3 ß (GSK3ß) at Ser9 and phosphorylated mammalian target of rapamycin complex 1 (mTORC1) levels in SAMP8 treated mice compared to SAMP8 control. Moreover, MCR5 treatment altered N-methyl-d-aspartate receptor (NMDA) 2B phosphorylation, and decreased the protein levels of phosphorylated cyclin-dependent kinase 5 (p-CDK5) and dopamine- and cyclic adenosine monophosphate (cAMP)-regulated phosphoprotein of Mr 32 kDa phosphorylated at Thr75 (p-DARPP32), with a parallel increase in protein kinase A (PKA) and p-cAMP response element-binding (pCREB) levels. Consistent with these changes MCR5 attenuated neuroinflammation by decreasing expression of pro-inflammatory markers such as Tumor necrosis factor-alpha (Tnf-α), Interleukin 1ß (Il-1ß), Interleukin 6 (Il-6), and promoted synaptic plasticity by increasing levels of postsynaptic density protein 95 (PSD95) as well as ameliorating tropomyosin-related kinase B (TrkB) and nerve growth factor receptor (NGFR) signalling. Collectively, these results increase the potential of highly selective I2-IR ligands as therapeutic agents in age-related BPSD and cognitive alterations.

Bicyclic α-Iminophosphonates as High Affinity Imidazoline I2 Receptor Ligands for Alzheimer's Disease.

Imidazoline I2 receptors (I2-IR), widely distributed in the CNS and altered in patients that suffer from neurodegenerative disorders, are orphans from a structural point of view, and new I2-IR ligands are urgently required for improving their pharmacological characterization. We report the synthesis and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of a new family of bicyclic α-iminophosphonates endowed with relevant affinities for human brain I2-IR. Acute treatment in mice with a selected compound significantly decreased Fas-associated protein with death domain (FADD) in the hippocampus, a key signaling mediator of neuroprotective actions. Additionally, in vivo studies in the familial Alzheimer's disease 5xFAD murine model revealed beneficial effects in behavior and cognition. These results are supported by changes in molecular pathways related to cognitive decline and Alzheimer's disease. Therefore, bicyclic α-iminophosphonates are tools that may open new therapeutic avenues for I2-IR, particularly for unmet neurodegenerative conditions.

Behavioral and Cognitive Improvement Induced by Novel Imidazoline I2 Receptor Ligands in Female SAMP8 Mice.

As populations increase their life expectancy, age-related neurodegenerative disorders such as Alzheimer's disease have become more common. I2-Imidazoline receptors (I2-IR) are widely distributed in the central nervous system, and dysregulation of I2-IR in patients with neurodegenerative diseases has been reported, suggesting their implication in cognitive impairment. This evidence indicates that high-affinity selective I2-IR ligands potentially contribute to the delay of neurodegeneration. In vivo studies in the female senescence accelerated mouse-prone 8 mice have shown that treatment with I2-IR ligands, MCR5 and MCR9, produce beneficial effects in behavior and cognition. Changes in molecular pathways implicated in oxidative stress, inflammation, synaptic plasticity, and apoptotic cell death were also studied. Furthermore, treatments with these I2-IR ligands diminished the amyloid precursor protein processing pathway and increased Aß degrading enzymes in the hippocampus of SAMP8 mice. These results collectively demonstrate the neuroprotective role of these new I2-IR ligands in a mouse model of brain aging through specific pathways and suggest their potential as therapeutic agents in brain disorders and age-related neurodegenerative diseases.